Alcohol abuse and alcoholism are significant health problems that affect over 17 million people and cost nearly $200 billion annually. It is likely tha a solution to these problems will result from a better understanding of alcohol effects on neuronal ion channels and the proteins that modulate them. The goal of the proposed research will be a significant step towards understanding the properties of alcohol-binding sites at an atomic level. We believe this knowledge will be essential for future design and selection of drugs that could reduce craving or addiction induced by alcohol. Specifically, we will study the sites fo alcohol binding in ligand- gated ion channels (LGICs), which include GABAaRs and GlyRs. Our homology modeling and experimental methods will provide 3-dimensional visualization of GABAaRs to increase our understanding of alcohol's action. This innovative approach combines cutting-edge computational and neuroscience techniques with molecular biology. We expect our results will have a significant impact on the broader class of alcohol- binding sites in other important receptors of the nervous system. Our Approach focuses on three aspects of alcohol-binding sites via three Specific Aims: Where are alcohol-binding sites; intra-subunit versus inter- subunit in LGICs (Aim 1), which specific residues or segments in LGICs mediate the effect of alcohol binding at these sites (Aims 1 and 2), and how do these sites modulate ligand binding (Aims 2 and 3). In Aim 1, Trudell and Bertaccini will build computational models of alcohol-binding sites in GABA receptors and design site-directed mutations to test the models. Harris and Howard, under a subcontract to the University of Texas, Austin, will test the function of these mutated receptors. We will iteratively refine the models the Trudell group will use the models to predict the effects of mutations; the Harris group will test if the models are consistent with experimental data; and the Trudell group will then modify the models to fit the new data. They will address this controversial question: What is the most important alcohol effect site in GABAaR? Is it Intra-subunit or Inter-subunit? They will also test the hypothesis that the GABAaR TM3 helix must rotate during activation in order to incorporate all recent experimental data. In Aim 2, the Trudell and Harris laboratories will recreate the alcohol-binding site from GABAaRs in the homologous but natively EtOH insensitive ion channel, GLIC, by determining which residues are specific to EtOH binding in GABAR and mutating these into their corresponding homologous positions within GLIC. In Aim 3, Trudell and Bertaccini will use three docking programs to investigate binding of alcohol analogs. Our investigators have proven accomplishment in alcohol research and possess the resources necessary to accomplish our Aims. Our proposal is responsive to both the NIAAA initiative in computational neuroscience and the NIH Roadmap: Bioinformatics and Computational Biology. These significant studies will provide essential knowledge needed to design alcohol-binding antagonists which could revolutionize treatment for alcohol abuse and dependence.